1. Field of the Invention
The present invention relates to a novel polyhydroxyalkanoate (hereinafter, sometimes abbreviated as PHA) and also to a method for manufacturing PHA very efficiently using a microorganism having capability to produce the PHA and accumulate it in bacterial bodies.
In addition, the present invention relates to a method for producing PHA using a substituted alkane derivative as a raw material.
2. Related Background Art
It has been reported so far that a variety of microorganisms produce poly-3-hydroxybutyric acid (hereinafter, sometimes abbreviated as PHB) and other PHAs and accumulate them in bacterial bodies (“Handbook of Biodegradable Plastic”, edited by Research Association of Biodegradable Plastic, NTS Co., Ltd., pp.178-197). These polymers as well as conventional plastics can be utilized for production of various products by melt processing or the like. Further, the polymers are biodegradable, and therefore they have an advantage of being completely degraded by microorganisms in nature, and causing no pollution by being left in natural environment unlike many conventional synthetic polymer compounds. Further, they are also excellent in biocompatibility and expected to be applied to a medical soft member or the like.
It has been known that such PHA produced by a microorganism may have a variety of compositions and structures depending on types of microorganism, culture medium composition, culture conditions and the like, and mainly from the viewpoint of improving physical properties of PHA, the study has been performed so far for controlling such composition and structure.
<<1>> First, the biosynthesis of PHAs which are obtained by polymerizing monomer units with a relatively simple structure such as 3-hydroxybutyric acid (hereinafter, sometimes abbreviated as 3HB) includes the followings.
For example, it is reported that Alcaligenes eutropus H16 strain (ATCC No. 17699) and its variants produce copolymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid in various composition ratios with a carbon source varied in their culturing (U.S. Pat. Nos. 4,393,167 and 4,876,331).
In U.S. Pat. No. 5,200,332, a method for producing copolymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid by making a microorganism of Methylobacterium sp., Paracoccus sp., Alcaligenes sp., or Pseudomonas sp. contact a primary alcohol having 3 to 7 carbons is disclosed.
In U.S. Pat. No. 5,292,860 and Japanese Patent Application Laid-Open No. 7-265065, it is disclosed that a two-component copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid is produced by culturing Aeromonas caviae using oleic acid and olive oil as carbon sources.
In Japanese Patent Application Laid-Open No. 9-191893, it is disclosed that a polyester having monomer units of 3-hydroxybutyric acid and 4-hydroxybutyric acid is produced by culturing Comamonas acidovorans IFO 13852 strain using gluconic acid and 1,4-butanediol as carbon sources.
Recently, studies on a PHA comprising a 3-hydroxyalkanoic acid having medium-chain-length (abbreviated as mcl) wherein the number of carbons is up to about 12 have been carried out energetically. The synthetic route of such PHAs can be roughly classified into two parts, specifically examples of which will be shown in the following (1) and (2).
(1) Synthesis Using β-Oxidation:
In U.S. Pat. No. 5,135,859, it is disclosed that a PHA having a monomer unit of 3-hydroxyalkanoic acid having 6 to 12 carbons is produced by supplying an acyclic aliphatic hydrocarbon as a carbon source to Pseudomonas oleovorans ATCC 29347 strain. In Appl. Environ. Microbiol, 58 (2), 746 (1992), it is reported that Pseudomonas resinovorans produces a polyester with monomer units of 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid (the amount ratio: 1:15:75:9) using octanoic acid as a sole carbon source, and further a polyester with monomer units of 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid (the amount ratio: 8:62:23:7) using hexanoic acid as a sole carbon source. Herein, it is considered that a monomer unit of a 3-hydroxyalkanoic acid having longer chain than that of a fatty acid as a material passes through the synthetic route of the fatty acid described in (2).
(2) Synthesis Using Fatty Acid De Novo Biosynthesis
In Int. J. Biol. Macromol., 16 (3), 119 (1994), it is reported that Pseudomonas sp. 61-3 strain produces a polyester with monomer units of 3-hydroxyalkanoic acids such as 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid, and of 3-hydroxyalkenic acids such as 3-hydroxy-5-cis-decenic acid and 3-hydroxy-5-cis-dodecenic acid using sodium gluconate as a sole carbon source.
By the way, the biosynthesis of PHA is usually performed by PHA synthase using “D-3-hydroxyacyl-CoA” as a substrate which is generated as an intermediate of various metabolic pathways in the cells.
Herein, “CoA” means “coenzyme A”. As described in the prior art of the above (1), when using a fatty acid such as octanoic acid, nonanoic acid or the like as a carbon source, it is said that the biosynthesis of PHA is carried out using “D-3-hydroxyacyl-CoA” as a starting material which is generated during the “β-oxidation pathway”.
The reactions until PHA is biosynthesized through “β-oxidation pathway” are shown below. 
On the other hand, as described in the prior art of the above described (2), when PHA is biosynthesized using saccharides such as glucose or the like, it is said that the biosynthesis is carried out using “D-3-hydroxyacyl-CoA” as a starting material converted from “D-3-hydroxyacyl-ACP” which is generated in the “fatty acid de novo biosynthesis”
Herein, “ACP” means “acyl carrier protein”.
By the way, any of PHAs synthesized in the above (1) and (2) as described above is PHA which comprises monomer units having an alkyl group in the side chain, i.e. “usual PHA”.
<<2>> However, when considering application of such PHAs produced by a microorganism to a wider range, e.g. as a functional polymer, PHAs (“unusual PHAs”) in which substituents other than an alkyl group are introduced into the side chain are expected to be extremely useful. Examples of the substituent include those containing an aromatic ring (such as a phenyl group, a phenoxy group, a benzoyl group or the like), an unsaturated hydrocarbon, an ester group, an aryl group, a cyano group, a halogenated hydrocarbon, an epoxide or the like. Of them, PHAs having an aromatic ring have been studied extensively.
(a) Those Containing a Phenyl Group or its Partially Substituted Form
In Makromol. Chem., 191, 1957-1965 (1990) and Macromolecules, 24, 5256-5260 (1991), it is reported that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-phenylvaleric acid as a unit using 5-phenylvaleric acid as a substrate.
Specifically, it is reported that Pseudomonas oleovorans produces 160 mg (31.6% of the dry weight to the bacterial body) per liter of a culture solution of a PHA comprising 3-hydroxyvaleric acid, 3-hydroxyheptanoic acid, 3-hydroxynonanoic acid, 3-hydroxyundecanoic acid and 3-hydroxy-5-phenylvaleric acid in a ratio of 0.6:16.0:41.1:1.7:40.6 as monomer units using 5-phenylvaleric acid and nonanoic acid as substrates (molar ratio of 2:1, total concentration of 10 mmol/L), and also this produces 200 mg (39.2% of the dry weight to the bacterial body) per liter of a culture solution of PHA containing 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxy-5-phenylvaleric acid in a ratio of 7.3:64.5:3.9:24.3 as monomer units using 5-phenylvaleric acid and octanoic acid as substrates (molar ratio of 1:1, total concentration of 10 mmol/L). It is considered that the PHAs in this report is synthesized mainly through the β-oxidation pathway because nonanoic acid and octanoic acid are used.
The relating description is in Chirality, 3, 492-494 (1991) besides the above where change in the physical properties of the polymer is recognized which is presumably caused by containing a 3-hydroxy-5-phenylvaleric acid unit.
In Macromolecules, 29, 1762-1766 (1996), it is reported that Pseudomonas oleovorans produces a PHA containing 3-hydroxy-5-(4′-tolyl)valeric acid as a unit using 5-(4′-tolyl)valeric acid as a substrate.
In Macromolecules, 32, 2889-2895 (1999), it is reported that Pseudomonas oleovorans produces a PHA containing 3-hydroxy-5-(2′,4′-dinitrophenyl)valeric acid and 3-hydroxy-5-(4′-nitrophenyl)valeric acid as units using 5-(2′,4′-dinitrophenyl)valeric acid as a substrate.
(b) Those Containing a Phenoxy Group or the Partially Substituted Form
In Macromol. Chem. Phys., 195, 1665-1672 (1994), it is reported that Pseudomonas oleovorans produces a PHA copolymer of 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-9-phenoxynonanoic acid using 11-phenoxyundecanoic acid as a substrate.
Also in Macromolecules, 29, 3432-3435 (1996), it is reported that using Pseudomonas oleovorans, a PHA comprising 3-hydroxy-4-phenoxybutyric acid and 3-hydroxy-6-phenoxyhexanoic acid as units is produced from 6-phenoxyhexanoic acid, a PHA comprising 3-hydroxy-4-phenoxybutyric acid, 3-hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-8-phenoxyoctanoic acid as units is produced from 8-phenoxyoctanoic acid, and a PHA comprising 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-7-phenoxyheptanoic acid as units is produced from 11-phenoxyundecanoic acid. Some of polymer yields in this report are excerpted as follows.
TABLE 1Carbon sourceDry cell weightDry polymerYield(alkanoate)(mg/L)weight (mg/L)(%)6-Phenoxyhexanoic acid95010010.58-Phenoxyoctanoic acid820901111-Phenoxyundecanoic acid1501510
In Japanese Patent Publication No. 2989175, a homopolymer comprising a 3-hydroxy-5-(monofluorophenoxy)pentanoate (3H5(MFP)P) unit or a 3-hydroxy-5-(difluorophenoxy)pentanoate (3H5(DFP)P) unit, a copolymer comprising at least 3H5(MFP)P unit or 3H5(DFP)P unit, Pseudomonas putida capable of synthesizing these polymers, and the invention relating to a method for manufacturing the above described polymers where Pseudomonas genus is used is disclosed.
These production is performed by the following “two-step culture”.
Culture time: 24 hours for the 1st step; and 96 hours for the 2nd step.
The substrates and polymers obtained in each step are shown as follows.
(1) Polymer obtained: 3-hydroxy-5-(monofluorophenoxy)pentanoate homopolymer
Substrate in the 1st step: citric acid, yeast extract
Substrate in the 2nd step: monofluorophenoxyundecanoic acid
(2) Polymer obtained: 3-hydroxy-5-(difluorophenoxy)pentanoate homopolymer
Substrate in the 1st step: citric acid, yeast extract
Substrate in the 2nd step: difluorophenoxyundecanoic acid
(3) Polymer obtained: 3-hydroxy-5-(monofluorophenoxy)pentanoate copolymer
Substrate in the 1st step: octanoic acid or nonanoic acid, yeast extract
Substrate in the 2nd step: monofluorophenoxyundecanoic acid
(4) Polymer obtained: 3-hydroxy-5-(difluorophenoxy)pentanoate copolymer
Substrate in the 1st step: octanoic acid or nonanoic acid, yeast extract
Substrate in the 2nd step: difluorophenoxyundecanoic acid.
It is said that as the effect, the polymer having a phenoxy group substituted with one or two fluorine atoms at the end of side chain can be synthesized by assimilating a medium chain fatty acid having a substituent, and the stereoregularity and water repellency can be provided while keeping good workability with a high melting point.
Compounds substituted with cyano and nitro groups other than such forms substituted with such a fluoro group have also been studied.
In Can. J. Microbiol., 41, 32-43 (1995) and Polymer International, 39, 205-213 (1996), it is reported that using Pseudomonas oleovorans ATCC 29347 strain and Pseudomonas putida KT 2442 strain, PHA comprising 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as a monomer unit is produced using octanoic acid and p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as substrates.
In these reports, since any polymer has an aromatic ring at the side chain of PHA which is different from general PHA having an alkyl group at the side chain, it will be advantageous to obtain polymers having the physical properties originated in them.
(c) PHA containing a cyclohexyl group in the monomer unit is expected to exhibit the physical properties of the macromolecule which is different from that of PHAs comprising usual aliphatic hydroxyalkanoic acid as units, and an example of the production by Pseudomonas oleovorans is reported (Macromolecules, 30, 1611-1615 (1997)).
According to this report, Pseudomonas oleovorans is cultivated in the culture medium wherein nonanoic acid and cyclohexylbutyric acid or cyclohexylvaleric acid coexist, and the resulting PHA comprises a unit containing a cyclohexyl group and a unit originated from nonanoic acid (each ratio is unknown).
For the yield etc., it is reported that the results shown in Table 2 were obtained by varying the ratio of cyclohexylbutyric acid and nonanoic acid in the condition of 20 mmol/L of the substrate concentration in total to cyclohexylbutyric acid.
TABLE 2Nonanoic acid:cyclohexylbutyricacidCDWPDWYieldUnit5:5756.089.111.8Nonanoic acid,cyclohexylbutyricacid1:9132.819.314.5Nonanoic acid,cyclohexylbutyricacidCDW: dry cell weight (mg/L), PDW: dry polymer weight (mg/L), Yield: PDW/CDW (%). 
However, this example shows that the yield of the polymer per culture solution is insufficient, and also that the PHA itself obtained is mixed with the aliphatic hydroxyalkanoic acid originated in nonanoic acid in the monomer unit.
<<3>> Further, as a new category, the study is performed not only on the change of the physical properties but also for producing PHA having an appropriate functional group on the side chain to create a new function utilizing the functional group.
For example, in Macromolecules, 31, 1480-1486 (1996) and Journal of Polymer Science: Part A: Polymer Chemistry, 36, 2381-2387 (1998), etc., it is reported that PHA comprising a unit having a vinyl group at the end of the side chain was synthesized, then epoxidated with an oxidizing agent resulting in the synthesis of PHA containing a highly reactive epoxy group at the end of the side chain.
Further, as a synthetic example of PHA comprising a unit which has a sulfide expected to be highly reactive others than the vinyl group, it is reported in Macromolecules, 32, 8315-8318 (1999) that Pseudomonas putida 27N01 strain produces a PHA copolymer of 3-hydroxy-5-(phenylsulfanyl)valeric acid and 3-hydroxy-7-(phenylsulfanyl)heptanoic acid using 11-phenylsulfanylvaleric acid as a substrate.
As described above, while different compositions and structures of PHAs produced by microorganisms are obtained by varying types of microorganisms and composition of the culture medium, conditions of the culture and the like used for their production, it is not said yet to be sufficient for the physical properties when considering application as plastic. In order to further increase the use extent of the PHA produced by microorganisms, it is important to examine improvement of the physical properties more widely, and therefore it is essential to develop and explore PHAs comprising monomer units having further various structures and their manufacturing methods and microorganisms which can produce the desired PHA efficiently.
Moreover, in a typical producing method of PHA consisting of giving a microorganism a chemically synthesized substituted fatty acid as a substrate, there are many cases where a significant limitation on a chemical synthesis is imposed depending on the type, number, position and the like of a substituent to be introduced, since the carboxyl group of the substituted fatty acid is an active group in a chemical reaction, or because of the active group, complex handling such as protection and deprotection of the carboxyl group in the reaction steps of the chemical synthesis is required, and chemical reactions over several steps in the process are often required. Therefore, there was difficulty of the synthesis on an industrially producing level or requirement for much time, troubles and costs for the synthesis.
On the other hand, if “unusual PHA” can be produced using a substituted alkane, which is more easily synthesized chemically than substituted fatty acid, as a material, it is assumed that the above problems could be solved.
For production of PHA from alkane derivatives which have been reported so far, there are only examples that the corresponding PHAs were biosynthesized by microorganisms using straight chain alkanes and alkenes (alkanes containing double bonds) (Appl. Environ. Microbiol., 54, 2924-2932 (1988)), chlorine-substituted alkanes (Macromolecules, 23, 3705-3707 (1990)), fluorine-substituted alkanes (Biotechnol. Lett., 16, 501-506 (1994)) and alkanes containing acetoxy residues (Macromolecules, 33, 8571-8575 (2000)) as starting materials, whereas there are no synthetic examples of the corresponding PHAs from an alkane having a residue containing an aromatic ring as a substituent reported.